The ADP-ribosylation factor (ARF)-related GTPase ARF-related protein bindsto the ARF-specific guanine nucleotide exchange factor cytohesin and inhibits the ARF-dependent activation of phospholipase D

ADP-ribosylation factor-related protein (ARP) is a membrane-associated GTPa se with remote similarity to the family of ADP-ribosylation factors (ARF). In a yeast two-hybrid screen designed to identify proteins interacting with ARP, we isolated a partial cDNA of the ARF-specific guanine nucleotide exc hange factor msec7-1/cytohesin encoding its N terminus and most of the See? domain (codons 1-200). ARP and ARP-Q79L (GTPase-negative ARP) exhibited a higher affinity to mSec7-1-(1-200) than ARP-T31N (nucleotide exchange-defec tive ARP) in the two-hybrid assay. Similarly, full-length [S-35]mSec7-1/cyt ohesin was specifically adsorbed to glutathione-Sepharose loaded with gluta thione S-transferase (GST)-ARP-Q79L, GST-ARP, or GST-ARP-T31N, the latter e xhibiting the lowest binding affinity. Overexpression of ARP-Q79L, but not of ARP-T31N, in COS-7 cells reduced the fluorescence from co-expressed gree n fluorescent protein fused with mSea7-1/cytohesin or mSec7-2/ARNO in plasm a membranes as detected by deconvolution microscopy, Recombinant ARP and AR P-Q79L, but not ARP-T31N, inhibited the phospholipase D (PLD) activity stim ulated by mSec7-2/ARNO and ARF in a system of isolated membranes. Furthermo re, transfection of HEK-293 cells with ARP or ARP-Q79L, but not ARP-T31N, i nhibited the muscarinic acetylcholine receptor-3 induced PLD stimulation an d translocation of ARF from cytosol to membranes. These data suggest that t he GTP-bound form of ARP specifically binds mSec7-1/cytohesin, and that ARP may be involved in a pathway inhibiting the ARF-controlled activity of PLD .